The splicing of pre-mRNA transcript coding regions leads to the production of a variety of key protein variants essential to many cellular processes in humans and complex organisms. To date, it is still unknown as to how the splicing machinery termed as the spliceosome correctly recognizes pre-mRNA splice sites. During the early stages of spliceosome assembly, an essential auxiliary protein factor, U2AF, binds to a polypyrimidine and 'AG' dinucleotide consensus sequence at the 3' splice site. This is a crucial step in splice site assembly as it commits pre-mRNA substrates to be spliced. U2AF is a heterodimer comprised of U2AF65 and U2AF35. The role of U2AF65 at the 3' splice site is to bind the polypyrimidine tract and recruit other splicing factors to the pre-mRNA where its interactions have been well characterized. Currently, it is known that U2AF35 contacts the 'AG' consensus site, however, the overall contributions of U2AF35 at the 3' splice site are not well known. The aims of this proposed research intend to address the following critical questions, still un-answered in the pre-mRNA splicing field: (i) what is the fundamental means of U2AF35 contacting the pre-mRNA at the correct 3' splice site? and (ii) what is the structural basis for this interaction? The significance of these questions relies upon the fact that splice site recognition by U2AF is a critical step in gene regulation, where aberrant regulation of splicing has been linked to various diseases in humans. Furthermore, the domains of U2AF35 that contact the 3' splice site 'AG' consensus sequence and the spatial arrangement of these interactions is currently unknown. In this proposed research we will use a variety of biophysical techniques to characterize U2AF35 in complex with RNA substrates including the 3' splice site 'AG' consensus sequence. Briefly, we will utilize fluorescence anisotropy, electrophoretic mobility shifts assays (EMSA) and site-directed mutagenesis to determine the affinity and specificity of U2AF35/RNA interactions. Surface Plasmon resonance (SPR) and isothermal titration calorimetry (ITC) methods will be used to provide a kinetic and thermodynamic description of these interactions. Using X-ray crystallography we will determine the structure of a U2AF35 variant bound to an oligonucleotide containing the 3' splice site 'AG' consensus sequence. Finally, we will collect small angle X- ray scattering (SAXS) data on full-length U2AF both in the presence and absence of RNA to provide the molecular envelop of U2AF/RNA interactions in solution.